5(6)-methyl substituted fluorescein derivatives

ABSTRACT

5(6)-methyl substituted fluorescein derivatives and a process for producing 5(6)-methyl substituted derivatives. Also provided are methods for these utilizing these derivatives as indicator reagents in assays for analytes, indicator reagents which comprise specific binding members attached to these derivatives and test kits which contain these derivatives.

This is a division of U.S. patent application Ser. No. 08/421,143 filedApr. 13, 1995 now U.S. Pat. No. 5,496,925.

BACKGROUND ART

This invention relates generally to fluorescein derivatives, and moreparticularly, relates to 5(6)-methyl substituted fluorescein derivativesand their production by the reduction of 5(6)-carboxyfluorescein to5(6)-methyl substituted fluorescein.

There are numerous fluorescein derivatives that have functional groupswhich are suitable for reaction with other molecules. Many of thesederivatives have been used commercially in the production of tracers foranalytical applications which range from the probing of cell functionsto the monitoring of the level of one or more drugs in physiologicaltest samples. See, for example, C. Dive et al., Molecular and CellularProbes 2:31 (1988); M. L. Graber et al., Anal. Biochem. 156:202 (1986);P. J. Brynes et al., U.S. Pat. No. 4,869,132; and N. Y. Wang et al.,European Patent Application Publ. No. EP264797 (1988). Examples of suchapplications include the Fluorescence Polarization Immunoassay (FPIA)for use on commercially available instruments, such as the Abbott AD_(x)® instrument and the Abbott TD_(x) ® instrument (available from AbbottLaboratories, Abbott Park, Ill.). These derivatives include 5- and6-carboxyfluorescein, 5- and 6-aminofluorescein and4'-aminomethylfluorescein (M. T. Shipchandler, et. al., Anal. Biochem.,162.89 (1987). Of these, the 5- and 6-aminofluorescein compounds are themost difficult with which to work, since the amino group is not verynucleophilic. This difficulty is an expected consequence of the aminogroup being directly bound to the deactivating aromatic ring. Thecompound 4'-aminomethylfluorescein (4'-AMF) was an earlier attempt tosolve this problem by adding a methylene group between the amino groupand the aromatic ring. This addition succeeded in restoring normal aminogroup reactivity of 4'-AMF. However some 4'-AMF derivatives have beenfound to be unstable under the conditions of long term storage inaqueous buffers. Possibly this instability is due to a retro-Mannichreaction or subsequent elimination of the amino group (H. O. House,Modern Synthetic Reactions, 2nd Ed., 1972, pp 654-660). Additionally,the preparation and purification of 4'-AMF is tedious.

Martin et. al., (Cytometry 12:184-187 1991!) report a preparation of5-chloromethyl fluorescein diacetate by the halogenation of5-methylfluorescein diacetate without including any experimentaldetails. Also, Peerce et. al. (J. Biol. Chem. 260 (10):6026-31 1985!)report a methyl substituted fluorescein derivative with the givenstructure: ##STR1## However, the experimental procedure reported byPeerce et al. would indicate that the compound was actually of thefollowing structure: ##STR2## Khanna et. al. (U.S. Pat. No. 4,439,356)disclose xanthene compounds of the following structures, but again offerno experimental details: ##STR3##

The reduction of 5(6)-carboxyfluorescein derivatives is not known.However, in general, carboxylic acid derivatives (i.e., free acids,metal salts, amides, esters, acid halides, etc.) may be reduced by avariety of methods. These methods have been reviewed extensively in theliterature. See, for example, H. C. Brown, Boranes in Organic Chemistry,Cornell University Press (1972); and C. F. Lane, "Reductions of OrganicCompounds with Diborane," Chemical Reviews 76:773-799 (1976). Also, thereduction of activated carboxylic acids to alcohols with sodiumborohydride has been described. K. Ramasamy et al., Synthesis: 42(1982).

The present invention solves the problem of amino reactivity at the 5 or6 position by adding a methylene group between it and the aromatic ring.Additionally, the novel processes employed results in the availabilityof novel and useful derivatives available for use in tracer synthesis,and for use in analytical assays.

SUMMARY OF THE INVENTION

The present invention provides fluorescein derivatives of the followingformulas: ##STR4## wherein R is selected from the group consisting ofhydroxy (--OH), and protected hydroxy (--OZ); and

wherein X is selected from the group consisting of hydroxy (--OH),protected hydroxy (--OZ), amino (--NH₂), protected amino (--NH_(m)Z'_(2-m)), thiol (--SH), protected thiol (--SZ"), a leaving group, aMichael acceptor, phosphoramidite, phosphonate and a linking group.

Hydroxyl groups are routinely protected as alkyl or aryl ethers(Z=alkyl, aryl, alkenyl), silyl ethers (Z=silyl), esters (Z=acyl),carbonates (Z=--C(═O)--O-alkyl, --C(═O)--O-aryl, --C(═O)--O-alkenyl) andcarbamates (Z=--C(═O)--NH-alkyl, --C(═O)--NH-aryl, --C(═O)--NH-alkenyl).Amino groups are routinely protected as carbamates (Z'=--C(═O)--O-alkyl,--C(═O)--O-aryl, --C(═O)--O-alkenyl), biscarbamates (m=0;Z'=--C(═O)--O-alkyl, --C(═O)--O-aryl, --C(═O)--O-alkeny), amides(Z'=--C(═O)-alkyl, --C(═O)-aryl, --C(═O)-alkenyl), cyclic imides (m=0;Z'=phthaloyl), N-benzyl derivatives (Z'=--CH.sub.(n) aryl.sub.(3-n),n=1-3), imine derivatives (Z'=═CH.sub.(n) alkyl.sub.(2-n), =CH.sub.(n)aryl.sub.(2-n) n=0-2), silyl derivatives (Z'=silyl), N-sulfenylderivatives (Z'=--S-aryl, --S--CH.sub.(n) aryl.sub.(3-n), n=0-3), andN-sulfonyl derivatives (Z'=--SO₂ -aryl, --SO₂ -alkyl). Thiol groups areroutinely protected as thioethers (Z"=--CH.sub.(n) aryl.sub.(3-n),n=1-3, aklyl), thioesters (Z"=acyl), thiocarbonates (Z=--C(═O)--O-alkyl,--C(═O)--O-aryl, --C(═O)--O-alkenyl), thiocarbamates(Z=--C(═O)--NH-alkyl, --C(═O)--NH-aryl, --C(═O)--NH-alkenyl), anddisulfides (Z"=--S-alkyl, aryl).

The present invention also encompasses X of the above formula being a"leaving group". Thus, when X is a leaving group in the presentinvention, it may be chosen from the group consisting of halide (X=--Cl,Br, --I), sulfonate ester (X=--OS(═O)₂ -alkyl, --OS(═O)₂ -aryl),activated amino (X=--N(alkyl)₃ +, N₂).

The present invention also encompasses X of the above formula being a"Michael acceptor". Thus, in the present invention X can be chosen fromthe group consisting of substituents of the formula --O--"MA" or--N(H)_(n) "MA", wherein "MA" is the Michael acceptor of the formula##STR5## wherein a, b and c independently can be hydrogen, alkyl, andaryl; and wherein U is chosen from --CH(═O), --C(═O)R, --C(═O)NH₂, --CN,--NO₂, --S(═O)R, ----S(═O)₂ R, and n is 0 or 1. A preferred Michaelacceptor is ##STR6## When X is a linking group it is of the formula--O--A--B or --N(H)_(n) --A--B,

wherein n =0 or 1 to satisfy the nitrogen valency; and

wherein A is a spacer group consisting of from 0 to 50 carbon atoms andheteroatoms, including not more than ten heteroatoms, arranged in astraight or branched chain, saturated or unsaturated, with the provisosthat not more than two heteroatoms may be directly linked in thesequence --O--A--B or --N(H)_(n) --A--B, the sequence --O--A--B or--N(H)_(n) --A--B cannot contain --O--O--, that in the sequence--N(H)_(n) --A--B, when n=0, then N--A taken together may comprise aring of not more that 6 atoms; and that branchings may occur only oncarbon atoms; and

wherein B is a reactive functional group selected from --C(═O)OH, --NH₂,--CH(═O), halo (--Cl, --Br, --I), sulfonate ester(OS(═O)₂ -alkyl,--OS(═O)₂ -aryl), Michael acceptor, phosphoramidite, and phosphonate.

The present invention also provides a process for preparing 5 (6)methylsubstituted fluorescein derivatives which comprises (a) protectingthe hydroxyls at the 3' and 6' positions of the following formulas IIIand IV, separately or as a mixture: ##STR7## to yield compounds of thefollowing formula V and VI: ##STR8## (b) reducing the carboxylic acidfunction of compounds of formulas V and VI such as to form compounds ofthe following formulas VII and VIII: ##STR9## (c) when prepared as amixture, separating the isomers of formulas VII and VIIIchromatographically such as to give the pure isomers of formulas VII andVIII;

(d) converting the hydroxyl moiety of the compounds of the formulas VIIand VIII to a leaving group, independently or as a mixture such as togive compounds of the formula IX or X: ##STR10## (e) displacing theleaving group X with a nucleophilic "pro" amino, "pro" thiol, or sodiumsulfhydride group such as to give compounds of the formulas IX and X:##STR11## wherein X"=a "pro" amino, "pro" thiol group, or thiol; (f)converting the X" groups in compounds of formula XI or XII such as togive compounds of the formulas XIII or XIV ##STR12## wherein X"'=NH₂ orSH; and (g) deprotecting the hydroxyl functions at positions 3' and 6'of the compounds of the formulas XIII and XIV such as to give thecompounds of the formulas XV or XIV: ##STR13## wherein X'"=NH₂ or SH.The present invention also provides a method for the preparation 5 (6)methyl-substituted fluorescein derivatives which comprises steps a-eabove and steps f and g in their inverse order.

The present invention also provides a method for the preparation 5 (6)methyl-substituted fluorescein derivatives which comprises steps a-c,and g above to produce compounds of the formula XVII or XVIII: ##STR14##

The terms nucleophilic, "`pro` amino" and "`pro` thiol" include thosenitrogen and sulfur containing nucleophiles which are readily convertedto amino or thiol groups. Nucleophilic "pro" amino groups are selectedfrom the group ##STR15## consisting of dicarboximides anions, e.g.phthalimide anion iminodicarbonates anions, e.g.,dibenzyliminodicarbonate anion ##STR16## sulfonamide anions: ⁻ N(H)_(n)(alkyl, aryl, alkenyl, acyl)_(2-n) S(═O)₂ (alkyl, aryl, alkenyl), n=0-2;sulfonimide anions: ⁻ N(S(═O)₂ (alkyl, aryl, alkenyl))₂ ; O-substitutedhydoxylamines: NH₂ O(alkyl, aryl, acyl); O--substituted hydroxamic acidanions: N(acyl)O(alkyl, aryl, acyl); azide anion: N₃ ⁻ ; cyanide anion:⁻ CN; isocyanate anion: ⁻ N═C═O; and isothiocyanate anion: ⁻ N═C═S.

"Pro" thiol groups include thiourea: H₂ NC(═S)NH₂ ; sodiumN,N-dimethylthiocarbamate; thiosulfate anion: S₂ O₃ ²⁻ ; disulfideanion: S₂ ²⁻. The present invention also provides for fluoresceinconjugates of the formula XIX or XX; ##STR17## wherein R is as definedabove and X is chosen from the group consisting of --N(H)_(n-), --O--,or --S--:

wherein n=0 or 1 to satisfy the nitrogen valency;

wherein A is a spacer group consisting of from 0 to 50 carbon atoms andheteroatoms, including not more than ten heteroatoms, arranged in astraight or branched chain, saturated or unsaturated, with the provisosthat not more than two heteroatoms may be directly linked in thesequence --X--A--M--Q , the sequence --X--A--M--Q cannot contain--O--O--, that when X=--N(H)_(n) - in the sequence --X--A--M--Q, whenn=0 then N--A taken together may comprise a ring of not more that 6atoms; and that branchings may occur only on carbon atoms;

wherein M is a linking group selected from >C(═O), --NH--, --O--C(═O)--,--N(H)--C(═O)--, --N(H)--C(═S)--, --S--, --P(═O)(O--)-- and ; ##STR18##and wherein Q is a conjugation partner.

The present invention also provides methods for detecting the presencean analyte in a test sample, wherein the method comprises the step ofcontacting the test sample with an indicator reagent which comprises acompounds of the formula XIX or XX.

In addition, the present invention provides test kits which contain anindicator reagent comprising a compound of Formula XIX or XX.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the process of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides fluorescein derivatives of the followingformulas: ##STR19## wherein R is selected from the group consisting ofhydroxy (--OH), and protected hydroxy (--OZ); and

wherein X is selected from the group consisting of hydroxy (--OH),protected hydroxy (--OZ), amino (--NH₂), protected amino (--NH_(m)Z'_(2-m)), thiol (--SH), protected thiol (--SZ"), a leaving group, aMichael acceptor, phosphoramidite, phosphonate and a linking group.

Protecting groups for hydroxyl, amino and thiol functional groups arewell known in the art (T. W. Greene, Protective Groups In OrganicSynthesis. John Wiley and Sons, New York, 1981). Hydroxyl groups areroutinely protected as alkyl or aryl ethers (Z=alkyl, aryl, alkenyl),silyl ethers (Z=silyl), esters (Z=acyl), carbonates (Z=--C(═O)--O-alkyl,--C(═O)--O-aryl, --C(=═O)--O-alkenyl) and carbamates(Z=--C(═O)--NH-alkyl, --C(═O)--NH-aryl, --C(═O)--NH-alkenyl). Aminogroups are routinely protected as carbamates (Z'=--C(═O)--O-alkyl,--C(═O)--O-aryl, --C(═O)--O-alkenyl ), biscarbamates (m=0;Z'=--C(═O)--O-alkyl, --C(═O)--O-aryl, --C(═O)--O-alkenyl ) amides(Z'=--C(═O)-alkyl, --C(═O)-aryl, --C(═O)-alkenyl), cyclic imides(Z'=phthaloyl), N-benzyl derivatives (Z'=--CH.sub.(n) aryl.sub.(3-n),n=1-3), imine derivatives (Z'=═CH.sub.(n) alkyl.sub.(2-n), ═CH.sub.(n)aryl.sub.(2-n) n=0-2), silyl derivatives (Z'=silyl), N-sulfenylderivatives (Z'=--S-aryl, --S--CH.sub.(n) aryl.sub.(3-n), n=0-3), andN-sulfonyl derivatives (Z'=--SO₂ -aryl, --SO₂ -alkyl). Thiol groups areroutinely protected

-as thioethers (Z"=--CH.sub.(n) aryl.sub.(3-n), n=1-3, aklyl),thioesters (Z"=acyl), thiocarbonates (Z=--C(═O)--O-alkyl,--C(--O)--O-aryl, --C(═O)--O-alkenyl), thiocarbamates(Z=--C(═O)--NH-alkyl, --C(═O)--NH-aryl, --C(═O)--NH-alkenyl), anddisulfides (Z"=--S-alkyl, aryl)

The present invention also encompasses X of the above formula being a"leaving group". A "leaving group" has been defined in the art asfollows:

"In a reaction in which a substrate molecule becomes cleaved, part of it(the part not containing the carbon) is usually called the leavinggroup." (J. March, Advanced Organic Chemistry, 2nd Ed.,McGraw-Hill,N.Y., 1977, p 187).

Most commonly, halides are the most useful leaving groups innucleophilic reactions, while alcohols, amines and thiols must beactivated prior to reaction. Alcohols are most commonly activated byconversion to sulfonate esters, or halides, or generated in situ by theMitsunobu reaction; amines by quarternization or diazotization. Thus,when X is a leaving group in the present invention, it may be chosenfrom the group consisting of halide (X=--Cl, Br, --I), sulfonate ester(X=--OS(═O)₂ -alkyl, --OS(═O)₂ -aryl), activated amino (X=--N(alkyl)₃₊,N₂). It should be recognized that one of ordinary skill in the art maydetermine other leaving groups which also can be utilized in the novelprocesses of the present invention.

The present invention also encompasses X of the above formula being a"Michael acceptor". A "Michael acceptor" has been defined in the art asfollows:

"The nucleophilic addition of enolate (or analogous) anions to thecarbon carbon double bond of α,β-unsaturated ketones, aldehydes nitrilesor carboxylic acid derivatives, a process known as the Michael reaction.The unsaturated compounds in the reaction, often called Michaelacceptors, may include any unsaturated system having a functional groupcapable of stabilizing the carbanionic intermediate. The Michaelacceptors may also add a variety of nucleophiles such as alcohols,thiols, and amines." H. O. House, Modern Synthetic Reactions, W. A.Benjamin, Inc., Menlo Park Calif., 1972, pp. 595-96.

Common functional groups which can activate a double bond to this kindof nucleophilic addition include, --CH(═O), --C(═O)R, --C(═O)NH₂, --CN,--NO₂, --S(═O)R, --S(═O)₂ R. Thus, the in the present invention, X canbe chosen from the group consisting of groups of the formula --O--"MA"or --N(H)_(n") MA", wherein "MA" is the Michael acceptor of the formula##STR20## wherein a, b and c independently can be hydrogen, alkyl, andaryl and wherein U is chosen from --CH(═O), --C(═O)R, --C(═O)NH₂, --CN,--NO₂, --S(═O)R, ##STR21## --S(═O)₂ R, and n is 0 or 1. A preferredMichael acceptor is X=

When X is a linking group it is of the formula --O--A--B or --N(H)_(n)--A--B:

wherein n=0 or 1 to satisfy the nitrogen valency; and

wherein A is a spacer group consisting of from 0 to 50 carbon atoms andheteroatoms, including not more than ten heteroatoms, arranged in astraight or branched chain, saturated or unsaturated, with the provisosthat not more than two heteroatoms may be directly linked in thesequence --O--A--B or --N(H)_(n) --A--B, the sequence --O--A--B or--N(H)_(n) --A--B cannot contain --O--, that in the sequence N(H)_(n)--A--B, when n=0, then N--A taken together may comprise a ring of notmore that 6 atoms; and that branchings may occur only on carbon atoms;and

wherein B is a reactive functional group selected from --C(═O)OH, --NH₂,--CH (═O), halo (--Cl, --Br, --I), sulfonate ester(OS(═O)₂ -alkyl,--OS(═O)₂ -aryl), Michael acceptor, phosphoramidite, and phosphonate.

The present invention also provides a process for preparing 5 (6)methyl-substituted fluorescein derivatives which comprises:

(step a) protecting the hydroxyls at the 3' and 6' positions ofcompounds of the following formulas III and IV, independently or as amixture: ##STR22## to yield compounds of the following formula V and VI:##STR23## (step b) reducing the carboxylic acid function of compounds offormulas V and VI such as to form compounds of the following formulasVII and VII: ##STR24## (step c) when prepared as a mixture, separatingthe isomers of formulas VII and VIII chromatographically such as to givethe pure isomers of formula VII and VIII;

(step d) converting the hydroxyl moiety of the compounds of the formulaVII and VIII to a leaving group, independently or as a mixture, such asto give compounds of the formula IX or X: ##STR25## (step e) displacingthe leaving group X with a nucleophilic "pro" amino, "pro" thiol, orsodium sulfhydride group such as to give ##STR26## wherein X"=a "pro"amino, "pro" thiol group, or thiol; (step f) converting the X" groups incompounds of formula XI or XII such as to give compounds of the formulasXIII or XIV: ##STR27## wherein X"'=NH₂ or SH; (step g) deprotecting thehydroxyl functions at positions 3' and 6' of the compounds of theformulas XIII and XIV such as to give the compounds of the formulas XVand XVI: ##STR28##

The present invention also provides a method for the preparation 5 (6)methyl-substituted fluorescein derivatives which comprises steps a-eabove and steps f and g in their inverse order.

The present invention also provides a method for the preparation 5 (6)methyl-substituted fluorescein derivatives which comprises steps a-c,and g above to produce compounds of the formula XVII and XVIII:##STR29##

The terms nucleophilic "`pro` amino" and "'pro thiol" are herein definedas; those nitrogen and sulfur containing nucleophiles known in the artto be readily converted to amino or thiol groups. Nucleophilic "pro"amino groups are commonly chosen from the group consisting ofdicarboximides anions, e.g. phthalimide anion ##STR30## iminodicarbonateanions, e.g., dibenzyliminodicarbonate anion ##STR31## sulfonamideanions: ⁻ N(H)_(n) (alkyl, aryl, alkenyl, acyl)_(2-n) S(═O)₂ (alkyl,aryl, alkenyl), n=0-2; sulfonimide anions: ⁻ N(S(═O)₂ (alkyl, aryl,alkenyl))₂ ; O-substituted hydoxylamines: NH₂ O(alkyl, aryl, acyl);O-substituted hydroxamic acid anions: ⁻ N(acyl)O(alkyl, aryl, acyl);azide anion: N₃ ⁻ ; cyanide anion: ⁻ CN; isocyanate anion: ⁻ N═C═O; andisothiocyanate anion: ⁻ N═C═S.

Commonly used "pro" thiol groups include thiourea: H₂ NC(═S)NH₂ ; sodiumN,N-dimethylthiocarbamate; thiosulfate anion: S₂ O₃ ²⁻ ; disulfideanion: S₂ ²⁻. It will be recognized that one of ordinary skill in theart may determine other "pro" amino and "pro" thiol groups which alsowould work in the novel processes of the present invention.

The present invention also provides for fluorescein conjugates of thefollowing formulas XIX or XX: ##STR32## wherein R is as defined aboveand X is chosen from the group consisting of --N(H)_(n) --, --O--, or--S--;

wherein n=0 or 1 to satisfy the nitrogen valency;

wherein A is a spacer group consisting of from 0 to 50 carbon atoms andheteroatoms, including not more than ten heteroatoms, arranged in astraight or branched chain, saturated or unsaturated, with the provisosthat not more than two heteroatoms may be directly linked in thesequence --X--A--M--Q, the sequence --X--A--M--Q cannot contain--O--O--, that when X=--N(H)_(n) - in the sequence --X--A--M--Q, whenn=0 then N--A taken together may comprise a ring of not more that 6atoms; and that branchings may occur only on carbon atoms;

wherein M is a linking group selected from >C(═O), --NH--, --O--C(═O)--,--N(H)--C(═O)--, --N(H)--C(═S)--, --S--, --P(═O)(O--)-- and ##STR33##and wherein Q is a conjugation partner.

The present invention also provides methods for detecting the presencean analyte in a test sample, wherein the method comprises the step ofcontacting the test sample with an indicator reagent which comprises acompounds of the formula XIX or XX.

The starting material, 5(6)-carboxyfluorescein, is available fromEastman Kodak, Rochester, N.Y. A preparation of 3',6'diacetyl-5(6)-carboxyfluorescein previously has been describederroneously as the pure isomer 3',6'-diacetyl-6-carboxyfluorescein, andlacks any characterization of the compound. See, J. W. Bruning et al. J.Immunological Method 33:33-44 (1980). The compounds 5-aminomethyl and5-bromomethylfluorescein recently have become commercially availablefrom Molecular Probes, Inc., Eugene Oreg. 97492.

An approximately 40:60 mixture of 5(6)-carboxyfluorescein, which existin both an open form and a closed form, was modified to increase itssolubility in organic solvents and to differentiate the two carboxylgroups, by treatment with acetic anhydride. Other means of accomplishingthis solubility may be known to those skilled in the art, and couldinclude the acylation of the hydroxyls with a different anhydride, acidhalide or active ester, or silylation with any number of silyl halides,or alkylation to give rise to suitable derivatives. Once so treated, the5(6)-carboxyfluorescein derivative is soluble in organic solventscommonly used for the reduction of carboxylic acid derivatives.

For example the procedure for the conversion of 5(6)-carboxyfluoresceinto the diacetylated species followed that of the unsubsfitutedfluorescein as described by W. R. Orndorff and A. J. Hemmer,"Fluorescein and Some of Its Derivatives", J. Amer. Chem. Soc.49:1272-1280 (1927). Briefly, this procedure involved reaction of5(6)-carboxyfluorescein in acetic anhydride at reflux with sodiumacetate; aqueous workup; and purification of the product mixture,3',6'diacetyl-5(6)-carboxyfluorscein, by column chromatography onsilica.

The remaining carboxylic acid can be reduced to the alcohol by treatmentwith borane-tetrahydrofuran complex, borane-dimethylsulfide complex orany ther reducing agent selective for carboxylic acids in the presenceof esters, over he course of one to two days. A preferred reducing agentis a boraneimethylsulfide complex.

Alternatively, the carboxylic acid can be converted into the mixedcarbonic nhydride with ethyl chloroformate or other active ester andreduced with sodium orohydride (NaBH₄) in buffered methanol THF (pH 6.0)for approximately five minutes at 0° C., to the alcohol.

For example 3',6'diacetyl-5(6)-carboxyfluorescein was converted to3',6'diacetyl-5(6)-hydroxyr

nethylfluorescein by both methods previously described herein.

The mixture of alcohols thus formed can be separated conveniently atthis stage by column chromatography on silica gel following methodsknown to those of ordinary skill in the art. For example3',6'diacetyl-5(6)-hydroxymethylfluorescein is separated into the. pureisomers 3',6'diacetyl-5-hydroxymethylfluorescein and3',6'diacetyl-6-hydroxymethylfluorescein.

The alcohol at this stage can be converted into any of several otherderivatives, as is well-known in the art. A preferred derivative wasobtained by converting the alcohol into an amine. This was accomplishedby converting the hydroxyl into a leaving group and displacing it with anucleophilic "pro" amino group. Preferred "pro" amino groups aredibenzyl iminodicarbonate and phthalimide. This is convenientlyperformed under the Mitsunobu conditions, described in O. Mitsuno, "TheUse of Diethyl Azodicarboxylate and Triphenylphoshpine in Synthesis andTransformation of Natural Products," Synthesis:1 (1981).

Briefly, this method involved reaction of3',6'diacetyl-5-hydroxymethylfluorescein or3',6'diacetyl-6-hydroxymethylfluorescein with diethylazodicarboxylate,triphenylphosphine, and dibenzyl iminodicarbonate or phthalimide intetrahydrofuran at room temperature under an inert atmosphere; followedby purification by chromatography on silica gel. The subsequentdeprotection (removal) of the acetyl groups with basic methanolcontaining catalytic dimethylaminopryridine, followed by hydrazinolysisof the phthalimide group or acid treatment of the iminodicarbonate groupwith HBr/acetic acid, gave the 5 or 6-aminomethylfluorescein as the freebase or as the hydrobromide salt, respectively.

The fluorescein derivatives of the present invention can be reacted witha variety of conjugation partners such as proteins, peptides, aminoacids, DNA-or RNA probe sequences, hormones, steroids, vitamins, drugs,functionalized polymers, both soluble and insoluble, haptens and otherchemicals such as polychlorinated biphenyls. The conjugates of thepresent invention have one of the following general structures offormulas XIX or XX: ##STR34## wherein R is as defined above and X ischosen from the group consisting of --N(H)_(n-), --O--, or--S--;

wherein n=0 or 1 to satisfy the nitrogen valency;

wherein A is a spacer group consisting of from 0 to 50 carbon atoms andheteroatoms, including not more than ten heteroatoms, arranged in astraight or branched chain, saturated or unsaturated, with the provisosthat not more than two heteroatoms may be directly linked in thesequence --X--A--M--Q, the sequence --X--A--M--Q cannot contain--O--O--, that when X=--N(H)_(n) -- in the sequence --X--A--M--Q, whenn=0 then N--A taken together may comprise a ring of not more that 6atoms; and that branchings may occur only on carbon atoms;

wherein M is a linking group selected from >C(═O), --NH--,--O--C(═O)-,----N(H)--C(═O)--, --N(H)--C(═S)--, --S--, --P(═O)(O--)--and; ##STR35## and wherein Q is a conjugation partner.

In the conjugates of the present invention, the chemical bonds betweenthe reactive functional group on the fluorescein derivative and theconjugation partner can be made by a number of methods. It frequently ispreferable to form amide bonds. For example, on the fluoresceinderivative of Formula I or II (X=--O--A--B or N(H)_(n) --A--B) when B isa carboxyl group, there are several ways to form a bond with aminogroups on a conjugation partner using a variety of methods known tothose skilled in the art.

Thus, for example, amide bonds are formed by first activating thecarboxylic acid moiety. of the fluorescein derivative by reaction withan activating reagent such as. 1,3-dicyclohexylcarbodiimide and anadditive such as N-hydroxysuccinimide. The activated form of thefluorescein derivative then is reacted with the conjugation partner.Alternatively, the carboxylic acid containing fluorescein derivative maybe converted, with or without isolation, into a highly reactive mixedanhydride, acyl halide, acyl imidazolide, or mixed carbonate and thencombined with the conjugation partner.

Any of these methods results in a derivative of the Formula XIX or XX(--X--A--M--Q) wherein M=--C(═O)--NH--, with the --C(═O)-- coming fromthe fluorescein derivative and the --NH-- from the conjugation partner.One of ordinary skill in the art will realize that there are manyreagents that can be used to form amide bonds other than those listedherein.

A fluorescein derivative with a terminal amine functionality can betransformed into a highly reactive N-hydroxysuccinimide urethane byreaction with N,N'-disuccinimidyl carbonate in a suitable solvent, suchas acetonitrile or dimethylformamide; The resultant urethane then isreacted with the amino groups on the conjugation partner.

A fluorescein derivative with a terminal aldehyde functionality can becoupled to the amino or hydrazide groups on the conjugation partner inthe presence of sodium cyanoborohydride, by reductive aminationaccording the methods known to those skilled in the art.

Alternatively, a fluorescein derivative containing a terminal alcoholgroup can be coupled to the conjugation partner by first reacting itwith phosgene or a phosgene equivalent, such as di- or triphosgene orcarbonyldiimidazole, resulting in the formation of a highly reactivechloroformate or imidazoloformate derivative (usually withoutisolation). The resultant active formate ester is then reacted with theconjugation partner.

Alternatively, a fluorescein derivative containing a terminal thiolgroup can be coupled to the conjugation partner containing a Michaelacceptor; or a fluorescein derivative containing a Michael acceptor canreact with a conjugation partner that has a thiol group.

Alternatively, a fluorescein derivative containing a terminalphosphoramidite or phosphonate group can be coupled to the conjugationpartner containing a hydroxyl, amino or thiol group.

The so-formed derivatives can be utilized as indicator reagents in avariety of immunoassays for the detection of analytes. "Analyte," asused herein, is the substance to be detected which may be present in thetest sample. The analyte can be any substance for which there exists anaturally occurring specific binding member (such as, an antibody), orfor which a specific binding member can be prepared. Thus, an analyte isa substance that can bind to one or more specific binding members in anassay. "Analyte" also includes any antigenic substances includinginfectious disease antigens such as viral, bacterial, fungal,rickettsial or other antigens and also cancer markers such as CEA, alsomacromolecules, haptens and/or their metabolites, antibodies, andcombinations thereof. As a member of a specific binding pair, theanalyte can be detected by means of naturally occurring specific bindingpartners (pairs) such as the use of intrinsic factor protein as a memberof a specific binding pair for the determination of Vitamin B12, or theuse of lectin as a member of a specific binding pair for thedetermination of a carbohydrate. The analyte can include a protein, apeptide, an amino acid, DNA or RNA probe sequences, a hormone, asteroid, a vitamin, a drug including those administered for therapeuticpurposes as well as those administered for illicit purposes, or otherchemicals such as polychlorinated biphenyls, a bacterium, a virus, andmetabolites of or antibodies to any of the above substances. The detailsfor the preparation of such antibodies and the suitability for use asspecific binding members are well-known to those skilled in the art.

Test samples which can be tested by employing a tracer of the presentinvention described herein include biological fluids such as human andanimal body fluids and non-biological fluids. Examples of biologicalfluids include whole blood, serum, plasma, milk, cerebrospinal fluid,urine, serum and bodily exudates as well as cell culture supernatants,and the like. Examples of non-biological fluids include water, sledge,etc. Other examples of materials which may be tested include those whichcontain cells, including both fixed and nonfixed surgical tissuespecimens, blood cells, bone marrow, tissue culture cells, and the like.

A "capture reagent", as used herein, refers to an unlabeled specificbinding member which is specific either for the analyte as in a sandwichassay, for the indicator reagent or analyte as in a competitive assay,or for an ancillary specific binding member, which itself is specificfor the analyte, as in an indirect assay. The capture reagent can bedirectly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

The "indicator reagent" comprises a signal generating compound, thetracer of the present invention, which is capable of generating ameasurable signal detectable by external means conjugated (attached) toa specific binding member for the analyte(s). "Specific binding member"as used herein means a member of a specific binding pair. That is, twodifferent molecules where one of the molecules through chemical orphysical means specifically binds to the second molecule. In addition tobeing an antibody member of a specific binding pair for for the analyte,the indicator reagent also can be a member of any specific binding pair,including either hapten-anti-hapten systems such as biotin oranti-biotin, avidin or biotin, a carbohydrate or a lectin ; acomplementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to an analyte as in a sandwich assay, to the capture reagent asin a competitive assay, or to the ancillary specific binding member asin an indirect assay.

Solid phases that can be used include a microparticles, both magneticand non-magnetic, or a mixture of magnetizable polymeric microparticleswith chemically or physically bound antigens or antibodies. Polymericmicroparticles with chemically or physically bound antigens orantibodies can be used according to the invention as capture phases in abinding reaction to make use of the fast diffusion rates of theseparticles in solution to yield rapid results. Microparticles that can beused according to this invention include polystyrene, carboxylatedpolystyrene, polymethylacrylate or similar particles with radius in therange of from about 0.1 to 20 μm. Magnetizable microparticles that canbe used preferably have ferric oxide or chromium oxide cores andpolystyrene, carboxylated polystyrene, polymethylacrylate coating. Apreferred separation method for these particles is the use of constantor pulsed magnets, washing said particles, and then suspending theseparated particles in a vessel where a signal can be generated anddetected. Yet other solid supports are known to those in the art andinclude the walls of wells of a reaction tray, test tubes, polystyrenebeads, nitrocellulose strips, membranes, glass, and others.

It is contemplated that the reagent(s) employed for the assay can beprovided in the form of a kit with one or more containers such as vialsor bottles, with each container containing a separate reagent such asindicator reagents which comprise the tracers of the present inventionas signal generating compounds.

Any assay format which employs a tracer as part of an indicator reagentas a signal generating compound can be utilized. Various examples ofassay formats are described herein, and all contemplated will employ thetracer of the present invention as part of the indicator reagent capableof generating a detectable signal. For example, a test sample which maycontain the analytes of interest, a mixture of probes for the analyteslabeled as described herein and a mixture of capture phases for theanalytes are incubated for a period of time and under conditionssufficient to allow optimal immunochemical binding reactions for theanalyte to take place. The capture phase then is separated and washed. Asubstrate specific to the tracer of the invention then is added. Afterthe enzyme/substrate reaction reaches an end point, the separatedreaction mixture is stopped. The presence of analyte is determined bydetecting the signal generated from the capture phase, and comparing theresult obtained to known positive and negative samples.

Ion capture procedures for immobilizing an immobilizable reactioncomplex with a negatively charged polymer, described in co-pending U.S.patent application Ser. No. 150,278 filed Jan. 29, 1988, which enjoyscommon ownership and is incorporated herein by reference, can beemployed according to the present invention to effect a fastsolution-phase immunochemical reaction. An immobilizable immune complexis separated from the rest of the reaction mixture by ionic interactionsbetween the negatively charged poly-anion/immune complex and thepreviously treated, positively charged porous matrix and detected byusing a tracer of the present invention to produce signal measurementsas described in co-pending U.S. patent application Ser. No. 425,643,which enjoys common ownership and is incorporated herein by reference.

It is contemplated that the indicator reagents of the present inventioncan be utilized in non-solid phase assay systems as well. For example, atest sample suspected of containing an analyte of interest is mixed witha labeled tracer and an antibody specific for the analyte and the traceris incubated for a time and under suitable conditions for a reaction tooccur. Any analyte which may be present in the test sample and thetracer compete for a limited number of binding sites on the antibody,resulting in the formation of analyte-antibody and tracer-antibodycomplexes. By maintaining constant the concentration of tracer andantibody, the ratio of the formation of analyte-antibody complex totracer-antibody complex is directly proportional to the amount ofanalyte present in the test sample.

Fluorescence-polarization methods of detection can be used as describedherein. Thus, the amount of analyte in the sample is determined byexciting the mixture with polarized light and measuring the polarizationof the fluorescence emitted by free tracer and treacer-antibodycomplexes. A tracer which is not complexed to an antibody is free torotate in less than the time required for adsorption and re-emission offluorescent light. As a result, the re-emitted light is relativelyrandomly oriented such that the fluorescence polarization of a tracernot complexed to an antibody is low, approaching zero. Upon complexingwith the specific antibody, the tracer antibody complex thus formedassumes the rotation of the antibody molecule, which is slower than thatof the relatively small tracer molecule, thereby increasing thepolarization observed. Therefore, when an analyte competes with thetracer for antibody sites, the observed polarization of fluorescence ofthe tracer-antibody becomes a value somewhere between that of the freetracer and the tracer-antibody complex. If the sample contains a highconcentraion of the analyte, the observed polarization value is closerto that of the free tracer, i,e, low. If the sample contains a lowconcentration of the analyte, the polarization value is closer to thatof the bound tracer, i.e., high. By sequentially exciting the reactionmixture of an immunoassay with vertically and then horizontallypolarized light, and analyzing only the vertical component of th emittedlight, the polarization of the fluorescence in the reaction mixture canbe accurately determined. The precise relationship between polarizationand concentration of the analyte to be determined is established bymeasuring the polarization values of calibrators having knownconcetrations. The concentration of the analyte can be interpolated fromthe standard curve prepared in this manner. This assay is referred to asa homogeneous assay, meaning that the final polarization readings aretaken from a solution in which bound tracer is not separated from freetracer.

When using fluorescence polarization, the results can be quantified interms of "millipolarization units," "span" (in millipolarization units)and "relative intensity." The measurement of millipolarization unitsindicates the maximum polarization when a maximum amount of the traceris bound to the antibody in the absence of any analyte in the testsample. The higher the net polarization units, the better the binding ofthe tracer to the antibody. The "span" is an indication of thedifference between the net millipolarization and the minimum amount oftracer bound to the antibody. A larger span provides for a betternumerical analysis of the data. The "relative intensity" is a measure ofthe strength of the fluorescence signal above the backgroundfluorescence. Thus, a higher intensity will give a more accuratemeasurement. The intensity is determined as the sum of the verticallypolarized lintensity plus twice the horizontally polarized intensity.The intensity can range from a signal of about three times to aboutthirty times the background noise, depending upon the concentration ofthe tracer and the other assay variables.Also, the pH at which thismethod is practised must be sufficient to allow the fluorescein moietyof the tracers to exist in their open form. The pH thus can range fromabout 4 to 9, preferably from about 6 to 8, and most preferably fromabout 7 to 7.5.Various buffers can be used to achieve and maintain thepH during the assay procedure. Representative buffers include borate,phosphate, carbonate, Tris, barbital and the like. The choice of aparticular buffer is not critical to the practice of this method.Generally, however, Tris and phosphate buffers are preferred.

The assay method described hereinabove is adaptable to be performed onautomated systems such as, but not limited to, the Abbott TD_(x) ®Therapeutic Drug Monitoring System, the Abbott AD_(x) ™ Abused DrugSystem, and the Abbott IM_(x) ® Fluorescence Polarization Analyzer, allof which are available from Abbott Laboratories, Abbott Park, Ill.60064.

The present invention will now be described by way of examples, whichare meant to illustrate, but not to limit, the spirit and scope of theinvention.

EXAMPLES Example 1

Step a: Acetylation of 5 (6) Carboxyfluorescein ##STR36##

5(6)-Carboxyfluorescein (10 g, 27 mmoles) was dissolved in aceticanhydride (200 mL) containing sodium acetate (4.4 g, 54 mmoles). Thesolution was heated to reflux for two (2) hours, allowed to cool to roomtemperature, and then poured into 1 L water. The mixture was stirredovernight at room temperature, after which the resulting precipitate wascollected by filtration, and air dried on the filter for 14 hours atroom temperature to give a dark brown solid product. The product wasfurther purified by chromatography on silica gel, eluting with ethylacetate. The resulting fractions which contained the pure product werecombined and evaporated to dryness under reduced pressure. The resultingsolid residue was recrystallized from ether/hexanes to give 7.8 g of3'-6'-diacetyl 5(6)-carboxyfluorescein.

Example 2

Step b,c: Reduction of 3'-6'-Diacetyl 5(6)-Carboxyfluorescein ##STR37##

3'-6'-Diacetyl 5(6)-carboxyfluorescein (0.5 g, 1.1 mmoles) was dissolvedin tetrahydrofuran. (THF) (5 mL), cooled to 0-5° C., and treated withtriethylamine (153 mL, 1.1 mmoles) and ethyl chloroformate (103 mL, 1.1mmoles) under a nitrogen atmosphere, to form a reaction mixture. Thereaction mixture was stirred for 1 hour, and then the reaction mixturewas filtered. The resulting filtrate was added to cold sodium phosphatebuffer (pH 6.0, 3 mL, 1M), then treated with sodium borohydride (83 mg,2.2 mmoles) in of water (1 mL). After 5 minutes, the reaction mixturewas diluted with water (50 mL) and extracted with ethyl acetate (50 mL).This extraction step was performed twice. The combined extracts weredried over sodium sulfate and evaporated to dryness to give 0.4 g of3'-6'-diacetyl 5(6)-hydroxymethylfluorescein. The mixture of isomers wasfurther purified and separated on silica gel (60 g), eluting with amixture of methylene chloride and methyl t-butyl ether at a ratio of9:1. The first eluting compound was3'-6'-diacetyl-6-hydroxymethylfluorescein. Calculated mass for C₂₅ H₁₉O₈ : 447.1080; found: 447.1075. The second eluting compound was3'-6'-diacetyl-5-hydroxymethylfluorescein. Calculated mass for C₂₅ H₁₉O₈ : 447.1080; found: 447.1075.

Example 3

Step d:Conversion of 3'-6'-diacetyl 5(6)-hydroxymethylfluorescein to5(6)-phthalimidomethylfluorescein

A mixture of 3',6'-Diacetyl 5(6)-hydroxymethylfluorescein (90 mg, 0.2mmoles) was dissolved in THF and treated with phthalimide (30 mg, 0.2mmoles), triphenylphosphine (53 mg, 0.2 mmoles) and diethylazodicarboxylate (DEAD) (32 mL, 0.2 mmoles) under a nitrogen atmosphereto form a reaction mixture. The reaction mixture was stirred for 30minutes, and then the reaction mixture was evaporated to dryness. Theresidue was chromatographed on silica (Chromatotron ™, HarrisonResearch, Palo Alto, Calif.), 2 mm plate, ethyl acetate and hexanes1:1!, at a rate of 10 mL/minute!. Further purification of the resultingproduct was accomplished by chromatography, by again eluting withmethylene chloride and ether (9:1) to give 3',6'- diacetyl5(6)-phthalimidomethylfluorescein (90 mg). ##STR38## Step e-g:Conversion of 3',6'- diacetyl 5(6)-phthalimidomethylfluorescein to5(6)-aminomethylfluorescein.

Example 4

Step e-g: Conversion of 3',6'- diacetyl5(6)-phthalimidomethylfluorescein to 5(6)-aminomethylfluorescein

3',6'- diacetyl 5(6)-phthalimidomethylfluorescein (90 mg) was dissolvedin methanol (1 mL) containing aqueous sodium hydroxide (0.1 N). Aftertwo (2) hours, hydrazine hydrate (100 mL) was added, and the resultingreaction mixture was stirred for another two (2) hours. At the end ofthat time, the reaction mixture was evaporated to dryness as describedpreviously in Examples 1 and 2. The residue was taken up in methanol (5mL) containing three drops of methanolic HCl (3N). Ether (50 mL) wasadded; and on cooling, 5(6)-aminomethylfluorescein precipitated as thehydrochloride salt and was collected by filtration. ##STR39##

Example 5

Preparation of 3',6'-diacetyl-6- (N-diCbz-amino)methyl!fluorescein##STR40##

3',6'-Diacetyl-6-hydroxymethylfluorescein (1 g, 2.2 mmol),triphenylphosphine (690 mg, 2.6 mmol), and dibenzyl iminodicarbonate(640 mg, 2.2 mmol) were stirred in THF (50 mL, from benzophenonepotassium ketyl) under a nitrogen atmosphere. To this solution was addeddiethyl azodicarboxylate (415 mL, 2.6mmol) in THF (25 mL) dropwise over30 min. After additional stirring for 1 h, the solution was evaporatedand the residue purified by filtration through silica gel by elutingwith methylene chloride/ether (9:1) to give 3',6'-diacetyl-6-(N-diCbz-amino)methyl!fluorescein (923 mg, 1.3 mmol, 59%). Calculatedmass for C₄₁ H₃₂ NO₁₁ : 714.1975; found: 714.1977.

Example 6

Preparation of 3',6'-diacetyl-5- (N-diCbz-amino)methyllfluorescein##STR41##

3',6'-diacetyl-5- (N-diCbz-amino)methyl!fluorescein was prepared from3',6'-diacetyl-5-hydroxymethylfluorescein in the same manner as Example5. (1 g, 1.4 mmol, 61%). Calculated mass for C₄₁ H₃₂ NO₁₁ : 714.1975;found: 714.1977.

Example 7

Conversion of 3',6'-diacetyl-6- (N-diCbz-amino)methyl!fluorescein to6-Aminomethylfluorescein

(A) 3',6'-diacetyl-6- (N-diCbz-amino)methyl!fluorescein (675 mg, 0.95mmol) from example 5, was stirred at reflux in methanol (50 mL)containing dimethylaminopyridine (10 mg) for 24 h. The solvent wasevaporated to give 6- (N-diCbz-amino)methyl!fluorescein. Calculated massfor C₃₇ H₂₈ NO₉ : 630.1764; found: 630.1763. ##STR42## (B) Withoutfurther purification 6- (N-diCbz-amino)methyl!fluorescein was dissolvedin methylene chloride (50 mL, from P₂ O₅) and treated with HBr/HOAc(31%, 5 mL). After stirring for 4 h the reaction mixture was filteredand the precipitate dried in vacuo for 12 h to give6-aminomethylfluorescein (410 mg, 0.93 mmol, 98%) as the hydrobromidesalt.

Calculated mass for C₂₁ H₁₆ NO₅ : 362.1028; found: 362.1027. ##STR43##

Example 8

Conversion of 3',6'-diacetyl-5- (N-diCbz-amino)methyl!fluorescein to5-Aminomethylfluorescein

(A) 5- (N-diCbz-amino)methyl!fluorescein was prepared from3',6'-diacetyl-5- (N-diCbz-amino)methyl!fluorescein (1 g, 1.4 mmol)example 6, by the procedure used in example 7A.

Calculated mass for C₃₇ H₂₈ NO₉ : 630.1764; found: 630.1763. ##STR44##(B) 5-Aminomethylfluorescein (600 mg, 1.36 mmol, 98%) was prepared from5- (N-diCbz-amino)methyl!fluorescein as in Example 7B. Calculated massfor C₂₁ H₁₆ NO₅ : 362.1028; found: 362.1027. ##STR45##

Example 9

Preparation of 5(6)-(maleimido) methylfluorescein ##STR46## (A)3'-6'-Diacetyl-5(6)-hydroxymethylfluorescein(100 mg, 0.2 mmoles),maleimide (20 mg, 0.2 mmoles), and triphenylphosphine (57 mg, 0.2mmoles) were dissolved in THF (10 mL). Diethylazodicarboxylate (40 μL,0.2 mmoles) was added. The reaction was stirred for 24 hours under anitrogen atmosphere. The reaction mixture was evaporated to dryness invacuo, and purified by chromatography (Chromatotron™, Harrison Research,Palo Alto, Calif.), 2 mm plate, methylene chloride and methyl t-butylether 9:1!, at a rate of 10 mL/minute!. to give3',6'-diacetytl-5(6)-(maleimido)methylfluorescein.

(B) 3',6'-Diacetytl-5(6)-(maleimido)methyl!fluorescein was treatedaccording to the procedure of Example 7A to give5(6)-(maleimido)methylfluorescein.

It is contemplated that the invention will be used for the preparationof fluorescein labeled haptens, proteins, nucleotides, nucleotides,oligonucleotides, polymers, solid phases, etc. 5-and6-Aminomethylfluorescein is a component of several FPIA tracers usefulin assays. One skilled in the art will appreciate the above-describedfluorescein derivatives and processes can be used under variedconditions to achieve optimum sensitivity and speed.

What is claimed is:
 1. A process for preparing 5(6) methyl-substitutedfluorescein derivatives which comprises:(a) protecting the hydroxyls atthe 3' and 6' positions of compounds of the following formulas III andIV independently or as a mixture: ##STR47## to yield compounds of thefollowing formula V and VI: ##STR48## wherein R is selected from thegroup consisting of hydroxy and protected hydroxy; (b) reducing thecarboxylic acid function of compounds of formulas V and VI such as toform compounds of the following formulas VII and VIII ##STR49## (c)deprotecting the hydroxyl functions at positions 3' and 6' of thecompounds of the formulas XIII and XIV so as to give the compounds ofthe formulas XVII or XVIII ##STR50##
 2. The process of claim 1 furthercomprising a mixture and wherein the isomers of formulas VII and VIIIare separated chromatographically after step (b) such as to give theisomers of said formula VII and VIII.
 3. The process of claim 1 furthercomprising performing the following steps after step (b) and before step(c), to yield compounds of formula XV or XVI in step (g), as follows:(i)converting the hydroxyl moiety of the compounds of the formulas VII andVIII to a leaving group, independently or as a mixture such as to givecompounds of the formula IX or X: ##STR51## (ii) displacing the leavinggroup X with a substituent selected from the group consisting ofnucleophilic "pro"amino, "pro" thiol, and sodium sulfhydride group suchas to give compounds of the formulas IX and X: ##STR52## wherein X"=a"pro" amino, "pro" thiol group, or thiol; (iii) converting the X" groupsin compounds of formula XI or XII such as to give compounds of theformulas XII or XIV ##STR53## wherein X"'=NH₂ or SH; and (c)deprotecting the hydroxyl functions at positions 3' and 6' of thecompounds of the formulas XIII and XIV so as to give the compounds ofthe formulas XV or XIV: ##STR54## wherein X"'=NH₂ or SH.
 4. The processof claim 2, further comprising performing the following steps afterseparating the isomers VII and VIII chromatagraphically:(i) convertingthe hydroxyl moiety of the compounds of the formulas VII and VIII to aleaving group, independently or as a mixture such as to give compoundsof the formula IX or X: ##STR55## (ii) displacing the leaving group Xwith a substituent selected from the group consisting of nucleophilic"pro" amino, "pro" thiol, and sodium sulfhydride group such as to givecompounds of the formulas IX and X: ##STR56## wherein X"=a "pro" amino,"pro" thiol group, or thiol; (iii) converting the X" groups in compoundsof formula XI or XII such as to give compounds of the formulas XIII orXIV ##STR57## wherein X"'=NH₂ or SH; and (c) deprotecting the hydroxylfunctions at positions 3' and 6' of the compounds of the formulas XIIIand XIV so as to give the compounds of the formulas XV or XIV: ##STR58##wherein X"'=NH₂ or SH.
 5. The process according to claim 3 or 4 whereinsaid nucleophilic "pro" amino groups are selected from the groupconsisting of dicarboximide anions, iminodicarbonate anions, sulfonamideanions, sulfonimide anions, O-substituted hydoxylamines, O-substitutedhydroxamic acid anions, azide anions, cyanide anions, isocyanate anionsand isothiocyanate anions.
 6. The process according to claim 5 whereinsaid dicarboximide anion is the phthalimide anion ##STR59##
 7. Theprocess according to claim 5 wherein said iminodicarbonate anion isdibenzyliminodicarbonate anion ##STR60##
 8. The process according toclaim 5 wherein said sulfonamide anions are --NH_(n) R'_(2-n) S(═O)₂ R",n is 0, 1 or 2,R' is an alkyl, aryl, alkenyl or acyl group, and R" is analkyl, aryl or alkenyl group;wherein said sulfonimide anions are--N(S(═O)₂ R")₂ ; wherein said O-substituted hydroxylamines are NH₂OR"', R"' is an alkyl, aryl or acyl group; and wherein saidO-substituted hydroxarnic acid anions are --N(R₁)OR"', R₁ is an acylgroup.
 9. The process according to claim 3 or 4 wherein said "pro"thiolgroups are selected from the group consisting of thiourea, sodiumN,N-dimethylthiocarbamate, thiosulfate anion and disulfide anion.